Employing Chimeric Xylanases to Identify Regions of an Alkaline Xylanase Participating in Fnzyme Activity at Basic pH(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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The xylanase A (XynA) from the alkaliphilic Bacillus halodurans C-125 and the xylanase B (XynB) from Clostridium stercorarium F9 were subdivided into four fragments at highly homologous regions present in their primary structures: an amino-terminal region (A or a), a region containing the putative proton donor (P or p), a region containing the putative catalytic nucleophile (N or n), and a carboxyl-terminal region (C or c). Six chimeric xylanases were constructed by the selective substitution of the four fragments using an overlapping PCR technique. Two of the six xylanases, APnc and Apnc (regions originating from XynA are denoted by upper case letters and those from XynB are denoted by lower case letters), were produced in Escherichia coli while the other four xylanases were obtained only as inclusion bodies. The APnc and Apnc chimeric enzymes were purified by column chromatography using Ni-NTA agarose and DEAE-Toyopearl. The respective pH and temperature stabilities of the purified enzymes were observed from pH 5.6 to 11.6 and up to 45℃ for APnc, and from pH 5.6 to 11.2 and up to 45℃ for Apnc. Thus, these enzymes were slightly less stable than the parental xylanases. An assessment of the pH-activity relationships for the chimeric xylanases employed p-nitrophenyl-β-D-xylobioside as the substrate in determinations of the k_cat values. The pK_a1, values for the APnc and Apnc chimeric enzymes were 4.3 and 4.2, respectively, which were almost identical to those for the parental xylanases. In contrast, the pK_a2 values obtained for APnc and Apnc were 9.1 and 8.5, respectively; these values fall between those for the parental xylanases, XynA (9.4) and XynB (7.8). These results indicate that the main regions necessary to maintain the high pK_a2 value of XynA locate in the A and P sections.
社団法人日本生物工学会の論文
2002-11-25

Employing Chimeric Xylanases to Identify Regions of an Alkaline Xylanase Participating in Fnzyme Activity at Basic pH(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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